UTR Mrna Isoform by CRISPR–Cas9 Gene Editing Impairs Dorsal Root Ganglion Development and Hippocampal Neuron Activation in Mice

Downloaded from rnajournal.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Elimination of Calm1 long 3′′′′′-UTR mRNA isoform by CRISPR–Cas9 gene editing impairs dorsal root ganglion development and hippocampal neuron activation in mice BONGMIN BAE,1,3 HANNAH N. GRUNER,1,3 MAEBH LYNCH,1 TING FENG,1 KEVIN SO,1 DANIEL OLIVER,2 GRANT S. MASTICK,1 WEI YAN,1,2 SIMON PIERAUT,1 and PEDRO MIURA1 1Department of Biology, University of Nevada, Reno, Nevada 89557, USA 2Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, Nevada 89557, USA ABSTRACT The majority of mouse and human genes are subject to alternative cleavage and polyadenylation (APA), which most often leads to the expression of two or more alternative length 3′′′′′ untranslated region (3′′′′′-UTR) mRNA isoforms. In neural tissues, there is enhanced expression of APA isoforms with longer 3′′′′′-UTRs on a global scale, but the physiological relevance of these alternative 3′′′′′-UTR isoforms is poorly understood. Calmodulin 1 (Calm1) is a key integrator of calcium signaling that generates short (Calm1-S) and long (Calm1-L)3′′′′′-UTR mRNA isoforms via APA. We found Calm1-L expression to be largely restricted to neural tissues in mice including the dorsal root ganglion (DRG) and hippocampus, whereas Calm1-S was more broadly expressed. smFISH revealed that both Calm1-S and Calm1-L were subcellularly localized to neural pro- cesses of primary hippocampal neurons. In contrast, cultured DRG showed restriction of Calm1-L to soma. To investigate the in vivo functions of Calm1-L, we implemented a CRISPR–Cas9 gene editing strategy to delete a small region encom- passing the Calm1 distal poly(A) site. This eliminated Calm1-L expression while maintaining expression of Calm1-S. Mice ΔL/ΔL lacking Calm1-L (Calm1 ) exhibited disorganized DRG migration in embryos, and reduced experience-induced neuronal activation in the adult hippocampus. These data indicate that Calm1-L plays functional roles in the central and peripheral nervous systems. Keywords: 3′′′′′-UTR; calmodulin; alternative polyadenylation; mRNA localization; axon; dorsal root ganglion; hippocampus INTRODUCTION neurons (Tushev et al. 2018). Thousands of genes in mouse and human express long 3′-UTR mRNA isoforms in brain Alternative cleavage and polyadenylation (APA) is the pro- tissues (Miura et al. 2013). For many genes, short 3′-UTR cess by which a pre-mRNA is cleaved and polyadenylated isoforms were found to be expressed across various tis- at two or more polyadenylation [poly(A)] sites. This com- sues, whereas the alternative long 3′-UTR isoforms were monly results in two (or more) mRNAs with the same pro- abundant only in brain. Our understanding of the in vivo tein coding sequence but different length 3′-UTR. APA functions of neural-enriched long 3′-UTR isoforms is limit- is pervasive, occurring in ∼51%–79% of mammalian genes ed despite the large number of genes shown to be affect- (Hoque et al. 2013; Lianoglou et al. 2013). The 3′-UTR is a ed in Drosophila, Zebrafish, mice, and humans (Smibert major target for posttranscriptional regulation via micro- et al. 2012; Ulitsky et al. 2012; Miura et al. 2013). RNAs (miRNAs) and RNA binding proteins (RBPs); thus, Multiple studies have examined the functions of 3′-UTRs harboring a longer 3′-UTR can theoretically confer addi- in vivo by generating mice that either genetically delete tional regulatory opportunities for transcripts (Sandberg parts of 3′-UTRs or introduce foreign poly(A) sites to cause et al. 2008; Mayr and Bartel 2009). Long 3′-UTRs impact production of mRNAs with truncated 3′-UTRs (Miller et al. translation in a cell context-specific manner (Floor and 2002; Perry et al. 2012). For example, a genetic approach Doudna 2016; Blair et al. 2017), and elements located in was implemented in mice to abolish the BDNF long 3′-UTR alternative 3′-UTRs can influence mRNA localization in isoform while maintaining expression of its short mRNA counterpart via the insertion of tandem SV40 poly(A) sites 3 These authors contributed equally to this work. Corresponding author: [email protected] Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna. © 2020 Bae et al. This article, published in RNA, is available under a 076430.120. Freely available online through the RNA Open Access Creative Commons License (Attribution 4.0 International), as described option. at http://creativecommons.org/licenses/by/4.0/. 1414 RNA (2020) 26:1414–1430; Published by Cold Spring Harbor Laboratory Press for the RNA Society Downloaded from rnajournal.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Loss of Calm1-L impairs neuronal function downstream from the proximal polyadenylation signal. has a specific function in the embryonic or adult nervous These mice displayed synaptic defects and hyperphagic system has not been addressed. obesity, ostensibly due to compromised mRNA localiza- Here, we utilized CRISPR–Cas9 to generate the first tion to dendrites and impaired translational control (An mouse lines lacking expression of a long 3′-UTR mRNA iso- et al. 2008; Liao et al. 2012). The recent advent of form while maintaining normal expression of its short 3′- CRISPR–Cas9 gene editing has revolutionized the speed UTR APA counterpart. We uncovered that Calm1-L levels and efficiency of generating deletion mouse strains were high in the DRG and hippocampus, and its expres- (Wang et al. 2013). This presents an exciting new opportu- sion was largely restricted to neurons. In vivo phenotypic nity for rapidly generating 3′-UTR isoform-specific knock- analysis of mutant embryos revealed disorganized DRG out mice. CRISPR–Cas9 has been implemented to axon and cell body positioning, whereas adults displayed successfully generate mice with a deletion that removed a reduction in hippocampal neuronal activation after most of the mTOR 3′-UTR (Terenzio et al. 2018). However, enriched environment exposure. This novel deletion meth- successful generation of an isoform-specific, long 3′-UTR odology has thus uncovered in vivo neuronal impairments knockout mouse using CRISPR–Cas9 has not been report- attributed to the loss of a single long 3′-UTR mRNA isoform ed to date. in mice. Calmodulin (CaM) is the primary calcium sensor in the cell (Means and Dedman 1980; Yamniuk and Vogel RESULTS 2004; Sorensen et al. 2013). CaM is expressed ubiquitous- ly but is particularly abundant in the nervous system Calm1-L expression is enriched in neurons (Kakiuchi et al. 1982; Ikeshima et al. 1993). There are three Calmodulin genes in mammals—Calm1, Calm2, and To determine the relative expression of short and long Calm3. These share an identical amino acid coding se- Calm1 3′-UTR isoforms among mouse tissues, we per- quence, but possess unique 5′ and 3′-UTRs (SenGupta formed northern blot analysis—an approach uniquely suit- et al. 1987; Fischer et al. 1988), suggesting differences in ed to report on the expression of alternative 3′-UTRs (Miura their regulation might be conferred at the posttranscrip- et al. 2014). Northern blots performed using a universal tional level. The existence of alternative 3′-UTR mRNA iso- probe (uni) that detects both short and long Calm1 forms generated by APA for the Calmodulin 1 (Calm1) 3′-UTR isoforms revealed the presence of both Calm1-L gene have been known for several decades (SenGupta and Calm1-S across an array of adult tissues. The cerebral et al. 1987; Nojima 1989; Ni et al. 1992; Ikeshima et al. cortex showed greater enrichment of Calm1-L versus 1993). These include mRNAs with a short 0.9 kb 3′-UTR Calm1-S compared to non-brain tissues (Fig. 1A,B), as (Calm1-S) and one with a long 3.4 kb 3′-UTR (Calm1-L). had been previously observed (Ikeshima et al. 1993; The functional significance of these alternative 3′-UTR iso- Miura et al. 2013). The ratio of long isoform to the sum forms is entirely unexplored. of long and short isoforms (total Calm1) was found to be Previous work has shown that altering CaM levels dis- 0.46 in cortex, whereas in the rest of the tissues examined rupts neuronal development in Drosophila and rodents (gastrocnemius skeletal muscle, heart, testes, kidney, and (Vanberkum and Goodman 1995; Fritz and VanBerkum liver) this value ranged between 0.12–0.28. Recent studies 2000; Kim et al. 2001; Kobayashi et al. 2015; Wang et al. have shown that some 3′-UTRs can be cleaved to generate 2015). CaM plays a role in guiding axon projections stable 3′-UTR transcripts that are separated from the pro- to create connections with other cells (Vanberkum and tein coding portion of the mRNA (Kocabas et al. 2015; Goodman 1995; Fritz and VanBerkum 2000; Kim et al. Malka et al. 2017; Sudmant et al. 2018). To test whether 2001; Kobayashi et al. 2015). Additionally, CaM interplays this might be the case for Calm1, and to confirm the con- with a variety of synaptic proteins to play a fundamental nectivity of the long 3′-UTR region to the rest of the Calm1 role in regulating signaling pathways critical in synaptic mRNA, the blot was stripped and reprobed with a probe plasticity (Xia and Storm 2005). Functions in the nervous specific for Calm1-L (ext). One band consistent with the system specifically for Calm1 have been described. size of Calm1-L was observed (Fig. 1C). Thus, the long Notably, targeted knockdown of Calm1, but not Calm2 3′-UTR region is connected to the full-length mRNA, with or Calm3, was found to cause major migration defects in no evidence of a cleaved 3′-UTR observed in the tissues developing hindbrain neurons (Kobayashi et al. 2015). examined. Calm1 mRNA has been detected in cultured rat embryonic We next determined the spatial expression pattern of dorsal root ganglion (DRG) axons, without distinguishing Calm1 3′-UTR isoforms in mouse tissues.

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